The similarity in staining between the elastic fibers and the fiber structure of FBXW2 tends to demonstrate that FBXW2 could be associated with elastic fibers (Figures S1 and S3). I previously reported that the cambium layer of the periosteum contains FBXW2 [17,18]. However, it was not known why FBXW2 showed a fiber-like structure. Figure 1a–c indicate the presence of microvessels and Figure 1c shows that FBXW2 is expressed in microvessels that are indicated with arrows but not in other microvessels. Figure 1d demonstrates that the expression pattern of elastin is similar to that of FBXW2. Figure 1e–g represent images of the periosteum, cambium layer, and bone, respectively. Elastin and FBXW2 were expressed in the same region in the elastic fibers. As shown in Figure 1 and Figure S1, FBXW2 was expressed in the elastic fiber-containing regions in bone and periosteum tissues. Double immunostaining of elastin leads to weak fluorescence signals at low magnification (Figure S3a). Therefore, single immunostaining was mainly employed. Moreover, double immunostaining did not quench the signal completely (Figure S2b). Different fibers with similar shapes, sizes, and regions, except for elastic fibers, might be present along with the elastic fibers. However, at high magnification, double immunostaining of elastin and FBXW2 revealed that the two proteins formed structural units of the fiber (Figure S3b–d). Elastic fibers are insoluble, and analysis of their components is difficult. In this study, FBXW2 and elastin were observed in similar regions in blood vessels. Therefore, FBXW2 might be associated with elastic fibers of the blood vessels. A comparison of FBXW2 and elastin localization within the periosteum indicated that some microvessels (indicated with arrows in the figures) contained both FBXW2 and elastin, whereas microvessels without elastin did not contain FBXW2. In large blood vessels, the expression patterns of elastin and FBXW2 were similar. Both elastin and FBXW2 within elastic fibers might play a role in supporting the blood vessel structure. Zhou et al. [22] studied lung cancer with FBXW2 as a suppressor, whereas Ren et al. [23] studied breast cancer with FBXW2 as a suppressor. Their studies focused on ubiquitination by FBXW2. However, to my knowledge, this is the first study on elastic fibers and FBXW2. FBXW2 has mainly been observed intracellularly and is known to induce ubiquitination [21]; it is unclear how FBXW2 is secreted into the extracellular space. Previously, I used two antibodies against FBXW2 (one from Abcam [18] and another from Invitrogen [17]). Antibodies against the synthetic peptide with sequence KRGSSFLAGEHPG, corresponding to the C terminal amino acids 410–422 in humans, were used, but at different periods with different labeled (peroxidase, alkaline phosphatase, and fluorescence) secondary antibodies [17,18]. These antibodies revealed the presence of FBXW2 in the extracellular space. In this study, both FBXW2 and elastic fibers were present in the tissue before the explant culture; moreover, cultured cells may not secrete FBXW2. Future studies should investigate how FBXW2 is secreted in the extracellular space (possibly by transportation with microvesicles or via unconventional protein secretion). Calcification was not observed in the blood vessels of 30-month-old cows. Previously, calcification was examined using alizarin red and von Kossa staining techniques to detect calcium deposits [24,25]. However, osteocalcin, an osteogenic differentiation marker, was examined to detect the calcification of vascular smooth muscle cells or vascular tissue [26,27,28,29,30]. In 2015, Hirashima et al. [31] reported that perforating fibers are present in the cambial layer, but the components of perforating fibers are still unclear. Elastic fibers may serve as the anchoring structure. The presence of FBXW2 in the periosteum and the relationship between FBXW2 and osteocalcin have been reported [18]; however, this study is the first to report the relationship between FBXW2 and elastic fibers.αSMA is expressed in vascular smooth muscle cells and myofibroblasts [32,33]. In this study, I observed that as αSMA-positive cells proliferated, the multi-layer of bovine PDCs became thick. αSMA might function as a native scaffold in PDCs. Previously, Uematsu et al. [34] compared the control medium (Medium 199) with MesenPRO-RS™ medium and reported that αSMA was expressed on the surface areas of periosteal sheets expanded in MesenPRO, but not on those cultured in the control medium. Notably, in the present study, the multi-layer of bovine PDCs cultured in Medium 199 was found to express αSMA. The observed differences may be because Uematsu et al. investigated periosteum from human alveolar bones, and the age of the subjects was not clear; whereas, in this study, I investigated periosteum from 30-month-old bovine legs.In this study, I also sought to determine whether calcification occurs around elastic fibers of microvessels in the periosteum. Elastic fibers are also contained in blood vessels. Under normal conditions, calcification of blood vessels rarely occurs. Elastic fibers in blood vessels do not contain osteocalcin (Figure 2e). Using double immunostaining, Akiyama [17] reported that FBXW2 and osteocalcin are co-expressed up to 7 weeks after explant culture. However, in this study, osteocalcin expression was not observed around small microvessels after explant culture for 6 and 7 weeks. Akiyama [18] reported a synthesized osteocalcin coat around the cord-like structure of FBXW2 during explant culture in the probable cambium layer of the periosteum. In this study, FBXW2 was expressed at the location of elastic fibers in the cambium layer of the periosteum and microvessels, whereas osteocalcin was expressed in the cambium layer, but not in microvessels. The different processes underlying the calcification of elastic fibers in the blood vessels and the cambium layer of the periosteum are still unknown. The periosteum contains osteogenic stem cells, whereas blood vessels do not. However, both tissues contain elastic fibers, FBXW2, and αSMA. A possible explanation is that FBXW2 in the cambium layer of the periosteum was uncovered, whereas FBXW2 in blood vessels was covered with endothelial cells and extracellular matrix, which made coating elastic fibers with osteocalcin difficult. FBXW2 was associated with elastin around elastic fibers in the cambium layer of the periosteum both before and during explant culture. Notably, Akiyama [18] reported solid fibers of FBXW2 and hollow fibers of osteocalcin. This finding can be explained by the fact that FBXW2 was present along with the elastic fibers and osteocalcin was expressed on these fibers.This study had some limitations. As the periosteum does not contain large blood vessels, the calcification processes of large blood vessels were not investigated in this study. Unlike the periosteum, the large blood vessels could not be placed on the culture dishes. Therefore, the calcification of large blood vessels remains to be elucidated. In this study, I used one condition of culture using Medium 199. In the outgrowth of PDCs and osteocalcin, differences between forelegs and hindlegs were not observed. Periosteum from the cranium or jaw was not tested. Akiyama [17] reported that osteocalcin synthesis occurs in FBXW2 fibers and hypothesized that the co-expression of osteocalcin and FBXW2 is related to osteogenic differentiation and bone formation. Therefore, during the explant culture of the periosteum, calcification for bone regeneration (such as osteocalcin expression) might occur around elastic fibers.

In conclusion, the calcification of elastic fibers in microvessels was not observed. However, at the same time, elastic fibers in the cambium layer calcified. To my knowledge, this study is the first to clarify the process of intramembranous ossification through elastic fibers of the cambium layer. Further studies on the roles of FBXW2 and elastic fibers may provide insights into and solutions to prevent the calcification of blood vessels.